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• W2074566613 abstract "Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member. Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member. Remarkable progress has been made in the past few years in understanding the molecular mechanisms of damage-induced mutagenesis. It has been suggested that a significant proportion of mutations arises when damaged genomic DNA is replicated in an error prone manner by one or more low fidelity polymerases (1Goodman M.F. Annu. Rev. Biochem. 2002; 71: 17-50Google Scholar). These polymerases appear to have evolved to specifically facilitate replication of a wide variety of DNA lesions that might otherwise block the high fidelity replication machinery. Most of these specialized polymerases are phylogenetically related to each other and have been collectively termed “Y-family” polymerases (2Ohmori H. Friedberg E.C. Fuchs R.P.P. Goodman M.F. Hanaoka F. Hinkle D. Kunkel T.A. Lawrence C.W. Livneh Z. Nohmi T. Prakash L. Prakash S. Todo T. Walker G.C. Wang Z. Woodgate R. Mol. Cell. 2001; 8: 7-8Google Scholar). The Y-family polymerases are ubiquitous and are found in all three kingdoms of life with many organisms often possessing more than one family member. The latter observations imply that Y-family polymerases play important roles in cellular survival or evolutionary “fitness” (3Friedberg E.C. Wagner R. Radman M. Science. 2002; 296: 1627-1630Google Scholar, 4Yeiser B. Pepper E.D. Goodman M.F. Finkel S.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8737-8741Google Scholar). Indeed, defects in human Polη 1The abbreviations used are: Pol, DNA polymerase; LF, little finger; CPD, cyclobutane pyrimidine dimer. result in the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant syndrome (5Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Google Scholar, 6Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Google Scholar), whereas mutations in Escherichia coli dinB reduce the ability of the cell to undergo adaptive mutagenesis in stationary phase (7McKenzie G.J. Lee P.L. Lombardo M.J. Hastings P.J. Rosenberg S.M. Mol. Cell. 2001; 7: 571-579Google Scholar, 8Tompkins J.D. Nelson J.L. Hazel J.C. Leugers S.L. Stumpf J.D. Foster P.L. J. Bacteriol. 2003; 185: 3469-3472Google Scholar). Although they share little primary amino acid sequence homology with DNA polymerases from other families, structural studies of two archaeal DinB-like polymerases (Dbh and Dpo4) and the catalytic core of Saccharomyces cerevisiae Polη reveal that they are topologically similar to classical polymerases in that they resemble a right hand and possess “fingers,” “palm,” and “thumb” subdomains. In addition they possess a unique domain that has been termed the “little finger” (LF) (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar), “wrist” (10Silvian L.F. Toth E.A. Pham P. Goodman M.F. Ellenberger T. Nat. Struct. Biol. 2001; 8: 984-989Google Scholar), or “PAD (polymerase associated domain)” (11Trincao J. Johnson R.E. Escalante C.R. Prakash S. Prakash L. Aggarwal A.K. Mol. Cell. 2001; 8: 417-426Google Scholar). The thumb and finger domains are smaller than those found in high fidelity polymerases and in the ternary complex of Dpo4 with DNA and an incoming nucleotide; the primer-template is held between the thumb and LF domains and buttresses against the finger domain (see Fig. 1A) (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar). The importance of the LF-DNA contact is highlighted by the fact that a proteolytic fragment of Dpo4 that retains the fingers, palm, and thumb subdomains (but lacks the LF domain) is much less active than the full-length polymerase (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar). Interestingly, the LF domain is the least conserved of the four domains in the Y-family polymerases, and it is hypothesized that such divergence may in part contribute to the assorted biochemical properties reported in the literature for the various Y-family polymerases (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar). To further investigate the role that the LF domain contributes to the overall enzymatic properties of Y-family polymerases, we have taken advantage of the fact that both structural and biochemical data are available for two closely related archaeal DinB-like polymerases, Dbh and Dpo4. Dbh (DinBhomolog) was identified and cloned by Kulaeva et al. (12Kulaeva O.I. Koonin E.V. McDonald J.P. Randall S.K. Rabinovich N. Connaughton J.F. Levine A.S. Woodgate R. Mutat. Res. 1996; 357: 245-253Google Scholar) in 1996 using degenerate PCR primers designed against the E. coli umuC and dinB genes. The genomic DNA used in those studies was from an archaeal strain obtained from the American Type Culture Collection (ATCC, Manassas, VA) that was originally believed to be Sulfolobus solfataricus P1. However, the entire genome of Sulfolobus acidocaldarius has recently been determined and the ∼2.5-kb dbh-containing sequence reported by Kulaeva et al. (12Kulaeva O.I. Koonin E.V. McDonald J.P. Randall S.K. Rabinovich N. Connaughton J.F. Levine A.S. Woodgate R. Mutat. Res. 1996; 357: 245-253Google Scholar) matches perfectly with the genomic sequence from S. acidocaldarius. 2R. Garrett, personal communication. Dbh therefore originates from S. acidocaldarius and not S. solfataricus P1 as was originally thought. Dpo4 (DNA polymerase IV) was identified in the genome of S. solfataricus P2 through BLAST searches (13Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Google Scholar) of the complete P2 genome (14She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-De Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. Van Der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Google Scholar), using the dbh gene as a search query (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar). Overall, the Dbh and Dpo4 proteins share 54% identity, yet the two polymerases exhibit different enzymatic properties (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar, 16Gruz P. Pisani F.M. Shimizu M. Yamada M. Hayashi I. Morikawa K. Nohmi T. J. Biol. Chem. 2001; 276: 47394-47401Google Scholar, 17Potapova O. Grindley N.D. Joyce C.M. J. Biol. Chem. 2002; 277: 28157-28166Google Scholar). Dpo4 is thermostable and exhibits robust polymerase activity. At high enzyme to template ratios Dpo4 can synthesize more than 1 kb of DNA, thereby allowing it to substitute for Taq polymerase in PCR assays (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar). In addition, the lesion bypass properties of Dpo4 are somewhat like those of the eukaryotic translesion polymerases in that Dpo4 can bypass thymine-thymine cyclobutane pyrimidine dimers (CPDs) (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar, 18Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Google Scholar, 19McCulloch S.D. Kokoska R.J. Masutani C. Iwai S. Hanaoka F. Kunkel T.A. Nature. 2004; 428: 97-100Google Scholar) and abasic sites (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar, 20Kokoska R.J. McCulloch S.D. Kunkel T.A. J. Biol. Chem. 2003; 278: 50537-50545Google Scholar). In contrast, Dbh is a much more distributive polymerase when replicating undamaged DNA, is unable to incorporate a base opposite a CPD, and bypasses an abasic site with very low efficiency (16Gruz P. Pisani F.M. Shimizu M. Yamada M. Hayashi I. Morikawa K. Nohmi T. J. Biol. Chem. 2001; 276: 47394-47401Google Scholar, 17Potapova O. Grindley N.D. Joyce C.M. J. Biol. Chem. 2002; 277: 28157-28166Google Scholar, 21Zhou B. Pata J.D. Steitz T.A. Mol. Cell. 2001; 8: 427-437Google Scholar). Structural studies of the two polymerases reveal that in addition to sharing high sequence homology, the fingers, palm, and thumb domains of the proteins are virtually superimposable. This suggests that the different enzymatic properties of the two enzymes might lie more in their sequence-divergent and structurally mobile LF domains. For example, in the Dpo4-DNA complex (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar), the linker connecting the thumb and little finger domains interacts only with DNA. In the apo-form of Dbh, however, this linker is hydrogen-bonded to the β-sheets in the palm domain as well as the β-sheets in the little finger domain, thereby pinning the little finger domain to the catalytic core (10Silvian L.F. Toth E.A. Pham P. Goodman M.F. Ellenberger T. Nat. Struct. Biol. 2001; 8: 984-989Google Scholar). For Dbh to bind substrate, this linker has to peel off from the palm domain to allow the little finger domain to reorient (see Fig. 1A). To investigate the role that the LF domain may play in determining the enzymatic properties of Y-family polymerases in general, we have constructed Dbh-Dpo4 chimeras in which the LF domains and the preceding linker have been interchanged (see Fig. 1B). Our studies reveal that by replacing the LF domain of Dpo4 with that from Dbh, we make the enzyme more “Dbh-like.” Conversely, by replacing Dbh LF with that of Dpo4, the enzyme becomes more “Dpo4-like,” indicating that the LF domain is clearly a major factor in determining the physical and enzymatic properties of each polymerase. We discuss our observations in light of the crystal structure of Dbh and of the various Dpo4-DNA complexes that have been reported to date. Overproduction of S. acidocaldarius Dbh—The dbh gene from S. acidocaldarius was PCR-amplified from pOS21 (12Kulaeva O.I. Koonin E.V. McDonald J.P. Randall S.K. Rabinovich N. Connaughton J.F. Levine A.S. Woodgate R. Mutat. Res. 1996; 357: 245-253Google Scholar) with two oligonucleotides: ssdbhbam (5′-CGC GGA TCC TTA AAT GTC GAA GAA ATC AGA TAA ATT TG-3′) and ssdbhbsp (5′-CAT GTC ATG ATA GTG ATA TTC GTT GAT TTT G-3′) containing a BamHI and BspHI restriction enzyme site, respectively (underlined). The ∼1050-bp PCR fragment was digested with BamHI and BspHI, and the fragment was gel-purified before cloning into pET16b (Novagen, Madison, WI) digested with NcoI and BamHI. The sequence of the dbh gene in the recombinant plasmid, called pJM349, was verified and subsequently introduced into E. coli strain RW382, a ΔumuDC595::cat derivative of BL21(λDE3) (22McDonald J.P. Frank E.G. Levine A.S. Woodgate R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1478-1483Google Scholar). Generation of Dbh-LF-Dpo4 and Dpo4-LF-Dbh Chimera—The first step toward generating Dbh/Dpo4 chimeras was to introduce a unique restriction enzyme site at the junction of the LF domain in Dpo4. This was achieved by site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) of Leu-228 (CTA → CTG) and Ala-229 (GCT → GCC) codons to produce a novel BalI restriction enzyme site within the dpo4 gene. The BalI restriction site was generated in the Dpo4 overexpressing plasmid, p1914 (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar), using oligonucleotides P2SWDW (5′-CTC GTC TCT GGC CAG AGA GAT CAA ATA TTT AGC C-3′) and P2SWUP (5′-TTG ATC TCT CTG GCC AGA GAC GAG TAT AAC GAG CC-3′) and gave rise to plasmid p1941. Chimeras were subsequently generated by domain swapping as follows. An ∼700-bp NdeI-BalI fragment was amplified using pJM349 (Dbh) DNA as a template with primers P1ndeIup (5′-GGG GGG CAT ATG ATA GTG ATA TTC GTT GAT-3′) and P1bal2dw (5′-GGG GGG ATT CTT GGC CAA CTT TAG TAG ATA TAA GGC TAA GGC-3′) containing NdeI and BalI restriction sites, respectively (underlined). The amplicon was then digested with NdeI and BalI and cloned into the similarly digested plasmid, p1941. The resulting plasmid, called p1947, therefore expresses a chimeric polymerase consisting of the thumb, finger, and palm domains of Dbh and the LF domain of the Dpo4 polymerase (Dbh-LF-Dpo4) (see Fig 1B). A second plasmid, p1946, expressing the thumb, finger, and palm domains of Dpo4 and the LF domain of the Dbh polymerase (Dpo4-LFDbh) (see Fig. 1B) was obtained by the amplification of a dbh fragment from pJM349 with oligonucleotides P1balIup (5′-GGG AAG TTG GCC AGA AAT AAA TAT AGT-3′) and P1bam2dw (5′-CCC CCC GGA TCC TTA AAT GTC GAA GAA ATC AGA-3′) containing BalI and BamHI sites, respectively (underlined). The amplicon was digested with BamHI and BalI and cloned into the similarly digested p1941 plasmid. The sequence of the chimeric dpo4LFdbh and dbhLFdpo4 genes in p1946 and p1947, respectively, were verified, and the plasmids were subsequently introduced into RW382. Purification of Dpo4, Dbh, Dbh-LF-Dpo4, and Dpo4-LF-Dbh Proteins—The protocol utilized to purify all four polymerases was based upon that described previously for Dpo4 (15Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Google Scholar) but includes several important modifications. Although all of the recombinant genes are under the control of an isopropyl-1-thio-β-d-galactopyranoside-inducible T7 promoter (in the parental pET vector), we found that there was significant expression of the recombinant proteins in the absence of induction. Furthermore, the Sulfolobaceae proteins are very stable in E. coli, 3F. Boudsocq, unpublished observations. and significant quantities of the recombinant proteins can be recovered by simply harvesting uninduced stationary phase overnight cultures of RW382 harboring the Dpo4/Dbh-expressing plasmids. Soluble cell extracts were made as described previously. In the heat denaturation step that removes significant quantities of the thermolabile E. coli proteins, the temperature was reduced from 85 to 75 °C. Each polymerase was purified to homogeneity in three chromatographic steps using HiTrapQ, hydroxylapatite, and Mono S columns as described previously except that the phosphate buffer used in the HiTrapQ column was replaced by a 20 mm HEPES buffer at pH 7.0 containing 100 mm NaCl, 1 mm dithiothreitol, and 0.1 mm EDTA. DNA Templates for in Vitro Primer Extension Assays—Most of the synthetic oligonucleotides used in the in vitro replication assays were synthesized by Lofstrand Laboratories (Gaithersburg, MD) using standard techniques and were gel-purified prior to use. Where utilized, the synthetic abasic site (dSpacer) was purchased from Glen Research (Sterling, VA) and was incorporated into oligonucleotide templates using standard protocols by Lofstrand Laboratories. The exception was the cis-syn cyclobutane pyrimidine dimer-containing oligonucleotide that was synthesized and purified by Phoenix Biotechnologies (Huntsville, AL). Primers were 5′-labeled with [γ-32P]ATP (5000 Ci/mmol; 1 Ci = 37 GBq) (Amersham Biosciences) using T4 polynucleotide kinase (Invitrogen). The sequence of each primer-template is given in the legend of the respective figure in which it was used. Single-stranded M13mp18 DNA was purchased from Invitrogen. In Vitro Primer Extension Assays—Radiolabeled primer-template DNAs were prepared by annealing the 5′-32P-labeled primer to the unlabeled template DNA at a molar ratio of 1:1.5. Standard 10-μl reactions contained 40 mm Tris·HCl at pH 8.0, 5 mm MgCl2, 100 μm each of ultrapure dNTP (Amersham Biosciences), 10 mm dithiothreitol, 250 μg/ml bovine serum albumin, 2.5% glycerol, and 10 nm primer-template DNA. The concentration of polymerase added varied and is given in Figs. 2, 3, 4, 5. After incubation at 37 or 60 °C for various times, reactions were terminated by the addition of 10 μl of 95% formamide, 10 mm EDTA, and the samples were heated to 100 °C for 5 min and were briefly chilled on ice. Reaction mixtures (5 μl) were subjected to polyacrylamide, 8 m urea gel electrophoresis, and replication products were visualized by PhosphorImager analysis.Fig. 3Processivity of Dpo4, Dbh, and the Dbh-LF-Dpo4 and Dpo4-LF-Dbh chimeras. Reactions were performed at 60 °C for 3 min in the presence of all four dNTPs (100 μm each) and contained 10 nm primer-template and limiting amounts of polymerase. The primer for these assays was a radiolabeled 23-mer (5′-GCG GTG TAG AGA CGA GTG CGG AG-3′) that was annealed to a 50-mer template (5′-CTC TCA CAA GCA GCC AGG CAA GCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′, where the location of the annealed primer is underlined). The concentration of enzyme in these reactions varied considerably and was determined empirically to allow us to compare the size distribution of replication products under conditions where the percentage of primers extended was comparable between the four enzymes. The concentrations of enzyme in the 10-μl reaction were as follows: Dbh, 0.2, 0.8, and 3.3 nm; Dbh-LF-Dpo4, 0.03, 0.17, and 0.83 nm; Dpo4, 0.017, 0.08, and 0.4 nm; Dpo4-LF-Dbh, 5.5, 7.7, and 11 nm. Based upon these assays, one can clearly see that both Dpo4 and Dbh-LF-Dpo4 are considerably more processive than either Dbh or Dpo4-LF-Dbh.View Large Image Figure ViewerDownload (PPT)Fig. 4Ability of the Dpo4, Dbh, and the Dbh-LF-Dpo4/Dpo4-LF-Dbh chimeras to replicate undamaged DNA and to bypass a synthetic abasic site or a cis-syn cyclobutane pyrimidine dimer. Reactions were performed at 60 °C for 5 min (undamaged DNA) or 10 min (abasic and CPD-templates) in the presence of all four dNTPs (100 μm each) and contained 10 nm primer template and 1, 10, or 100 nm of enzyme. The local sequence context is given at the left side of each panel. A, undamaged DNA; B, abasic site-containing DNA; C, CPD-containing DNA. The complete sequence of the undamaged template was 5′-CTC TCA CAA GCA GCC AGG CAT-TCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′. The 50-mer cis-syn dimer-containing template was identical except that it contained a single CPD located at the adjacent T letters indicated in bold typeface. The 50-mer abasic (X)-containing template was 5′ CTC TCA CAA GCA GCC AGG CAT XCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′. All three templates were primed with a radiolabeled 23-mer oligonucleotide with the following sequence: 5′-GCG GTG TAG AGA CGA GTG CGG AG-3′. Replication products were separated on a 12%/8 m urea polyacrylamide gel and replication products were visualized by PhosphorImager analysis.View Large Image Figure ViewerDownload (PPT)Fig. 5Specificity of Dpo4-, Dbh-, Dbh-LF-Dpo4-, and Dpo4-LFDbh-dependent nucleotide incorporation on an undamaged template. Standard 10-μl reactions were performed at 37 or 60 °C for 2 min and contained a 10 nm concentration of radiolabeled primer-template (primer, 5′-GTG TCG GGG CGA GTG CGC CG-3′; template, 5′-CTC TCA CAA GCA GCT AAG CAG CGG CGC ACT CGC CCC GAC ACC GC-3′, with the position of the annealed primer underlined) and various amounts of polymerase. Reactions performed at 37 °C (A) contained 30 nm Dbh, 75 nm Dpo4-LF-Dbh, 5 nm Dpo4, and 10 nm Dbh-LFDpo4. Those performed at 60 °C (B) contained 10 nm Dbh, 25 nm Dpo4-LF-Dbh, 2.5 nm Dpo4, or 5 nm Dbh-LF-Dpo4. Products were resolved by denaturing polyacrylamide gel electrophoresis (8 m urea, 15% acrylamide) and subsequently visualized using an Amersham Biosciences PhosphorImager.View Large Image Figure ViewerDownload (PPT) Forward Mutation Assay—Reaction mixtures (30 μl) contained 1 nm gel-purified M13mp2 gapped DNA substrate, 40 mm Tris·HCl (pH 9.0 at 22 °C), 5 mm MgCl2, 10 mm dithiothreitol, 7.5 μg of bovine serum albumin, 2.5% glycerol, and 1 mm concentration each of dATP, dGTP, dCTP, and dTTP. Polymerization reactions were initiated by adding 20 nm Dpo4-LF-Dbh or 1.5 nm Dbh-LF-Dpo4 incubated at 70 °C for 1 h and were terminated by adding EDTA to 15 mm. DNA products were analyzed by agarose gel electrophoresis and assayed for the frequency of lacZ mutants as described (23Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Google Scholar, 24Kokoska R.J. Bebenek K. Boudsocq F. Woodgate R. Kunkel T.A. J. Biol. Chem. 2002; 277: 19633-19638Google Scholar). DNA samples from independent lacZ mutant phage were sequenced to identify the sequence changes generated during gap-filling synthesis. Error rates were calculated as described previously (23Bebenek K. Kunkel T.A. Methods Enzymol. 1995; 262: 217-232Google Scholar, 24Kokoska R.J. Bebenek K. Boudsocq F. Woodgate R. Kunkel T.A. J. Biol. Chem. 2002; 277: 19633-19638Google Scholar). Generation of Little Finger Domain Chimeras—Native Dbh is a 354-amino acid protein with an estimated pI of 9.37. Dpo4 is two amino acids shorter and has an estimated pI of 9.11. Alignment of the two primary amino acid sequences reveals that although both proteins originate from related Sulfolobaceae, they share only 54% identity overall. Interestingly, most identity is found in the fingers, palm, and thumb subdomains of the polymerases, which are 59% identical. In contrast, the LF domain is least conserved, with only 41% primary amino acid sequence identity (Fig. 1B). To investigate the role that the LF domain plays in the enzymatic properties of Y-family polymerases, we constructed chimeric proteins in which the respective LF domains and the flexible linker that tethers the LF domain to the thumb domain were interchanged (Fig. 1B). The first step of the process was to introduce a novel BalI restriction site into the dbh and dpo4 genes at the site that corresponds to the very end of the “K” helix in the thumb domain of each polymerase (Fig. 1B) (18Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Google Scholar). In Dpo4 this is at the Ala-229 codon, whereas in Dbh the corresponding site is at Ala-230. The LF domain was then PCR-amplified using primers containing the novel BalI restriction enzyme site. After digestion with BalI, the amplicons were cloned into the appropriately digested parental vectors to make chimeras containing the fingers, palm, and thumb domains of Dpo4 and the LF of Dbh (termed Dpo4-LF-Dbh) or one containing the fingers, palm, and thumb domains of Dbh and the LF or Dpo4 (termed Dbh-LF-Dpo4). The LF domain of Dpo4 is 1 amino acid shorter than the Dbh LF domain, and as a consequence both chimeras are 353 amino acids long (Fig 1B). Size Distribution of Replication Products Synthesized by Native and Chimeric Dpo4 and Dbh Polymerases—In vitro replication reactions with Y-family polymerases have clearly established that they are less processive than high fidelity replicative polymerases. However, the absolute number of nucleotides incorporated per DNA binding event varies considerably among Y-family polymerases. For example, recent studies suggest that archaeal Dpo4 is more processive than human Polη (20Kokoska R.J. McCulloch S.D. Kunkel T.A. J. Biol. Chem. 2003; 278: 50537-50545Google Scholar). Indeed, when replicating circular M13 DNA at high enzyme to template ratios, Dpo4 synthesizes replication products that are several hundred nucleotides in length (Fig. 2). Under the same assay conditions, Dbh-dependent replication products are much shorter. Moreover, in contrast to Dpo4, adding a large molar excess of Dbh to the reaction does not dramatically change the size distribution of replication products on the circular M13 primer-template. Interestingly, the size distribution of replication products appears to be largely dependent upon the LF domain. Replacing the native LF domain of Dbh with that of Dpo4 leads to a dramatic increase in the size of the overall length of the replication products. Conversely, replacing the native LF domain of Dpo4 with that of Dbh reduces the size distribution of replication products from several hundred nucleotides at a 20-fold molar excess to ∼50 nucleotides or less at the same enzyme to template ratio (Fig. 2). Similar results were obtained in experiments performed at 60 °C with a shorter linear DNA template and a large molar excess of substrate over enzyme, which made it possible to more accurately measure the processivity of each enzyme during a single extension reaction (Fig. 3). Under reaction conditions where primer usage is minimal, full-length replication products are only observed in the presence of Dpo4 and the chimeric Dbh-LF-Dpo4, whereas those generated by either Dbh or Dpo4-LF-Dbh, are considerably shorter. Based upon these observations, we conclude that the respective LF domain of Dpo4/Dbh is the major factor determining the overall processivity of the two enzymes. Such conclusions are consistent with the crystallized ternary structure of Dpo4-DNA and incoming nucleotide, which revealed that the LF domain of Dpo4 in conjunction with the thumb domain wraps around DNA and helps hold the polymerase on to the primer terminus (Fig. 1A) (9Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Google Scholar). Effect of LF Domain Swapping on Translesion DNA Synthesis of a CPD and Abasic Site—Previous studies have shown that although Dpo4 is phylogenetically located in the DinB branch of the Y-family polymerases, it actually has enzymatic properties that are reminiscent of Polη-like enzymes in that it can bypass cis-syn cyclobutane pyrimidine dimers. The efficiency of a Dpo4-dependent bypass of a CPD has recently been estimated to be approximately one-tenth of that of human Polη (19McCulloch S.D. Kokoska R.J. Masutani C. Iwai S. Hanaoka F. Kunkel T.A. Nature. 2004; 428: 97-100Google Scholar). The reduced ability of Dpo4 to bypass a CPD compared with Polη appears to be largely caused by stearic clashes between the 5′-T of the CPD and Dpo4 when the enzyme attempts to incorporate a nucleotide opposite the covalently linked 3′-T of the CPD (18Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Google Scholar). Nevertheless, the ability of Dpo4 to bypass a CPD is greater than that of the related PolIV (25Tang M. Pham P. Shen X. Taylor J.-S. O'Donnell M. Woodgate R. Goodman M. Nature. 2000; 404: 1014-1018Google Scholar), Polκ (26Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3838-3843Google Scholar, 27Ohashi E. Ogi T. Kusumoto R. Iwai S. Masutani C. Hanaoka F. Ohmori H. Genes Dev. 2000; 14: 1589-1594Google Scholar, 28Zhang Y. Yuan F. Wu X. Wang M. Rechkoblit O. Taylor J.S. Geacintov N.E. Wang Z. Nucleic Acids Res. 2000; 28: 4138-4146Google Scholar), or Dbh polymerases (Fig. 4), which have little ability to incorporate a base opposite the 3′-T of the dimer. Likewise, Dpo4 can bypass a synthetic abasic site (15Boudsocq F. Iwai S. Hanaoka F. Wood" @default.
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• W2074566613 title "Investigating the Role of the Little Finger Domain of Y-family DNA Polymerases in Low Fidelity Synthesis and Translesion Replication" @default.
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